Mitochondria play a central role in determining whole organism fitness, but we have little information on the role of the mitochondrial genome in higher eukaryotes due in part to the difficulty of isolating viable mutants. In this project several classes of putative mitochondrial mutations in a higher eukaryote are described; these mutations confer extreme changes in whole organism phenotype: male sterility and fungal toxin susceptibility. These mutant classes are defined by the extent to which specific nuclear genes will restore fertility. Each mutant class shows maternal inheritance as expected for a mitochondrial mutation; each class can be bred into a uniform, highly inbred nuclear background. The DNA of mitochondria of each class will be isolated and analyzed as to contour length, denaturation pattern restriction enzyme digestion pattern, transcriptional products by RNA: DNA hybridization, and homology with normal mtDNA by heteroduplex analysis. A cleavage map will be constructed for each mtDNA type and mutations conferring sterility and toxin susceptibility mapped. The phenotype of each mitochondrial type will also be determined by examining enzyme polymorphism, membrane protein variation, ultrastructure, and reaction with toxin. The site and mode of action of the terpenoid fungal toxin will be determined with 3H and 14C in vivo labeled toxin; the toxin is a promising agent for use as a probe of membrane structure. This project will (a) provide an estimate of the existing variation in mitochondrial phenotype and genotype, (b) examine the co-operation between nuclear and cytoplasmic genomes in specifying an essential organelle, and (c) determine the sites of mitochondrial mutation conferring sterility and toxin sensitivity.